Active and Poised Promoter States Drive Folding of the Extended Hoxb

© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. addressed addressed to A.P. ( London, UK. London, Hammersmith Hospital Campus, London, UK. College London, Hammersmith Hospital Campus, London, UK. of Health (BIH), Berlin, Germany. Germany. 1 atedexchange with higher cells, of histone linker H1 and nucleosome differenti of chromatin the than dynamic more is chromatin cell ES cells differentiated than matin loci. Polycomb-repressed in regard to particularly with and regions active populated unknown, remains regions of folding more gene-dense complex, large-scale the into Polycomb-repressed domains in gene-poor areas mechanisms to loop-extrusion chromatin domains organize through chromatin of loops and enhancer–promoterformation interactions the in roles important has binding CTCF domains. chromatin Polycomb-repressed from active partition that configurations folding chromatin cell-type-specific establish to help may states activation gene that suggest unclear, they remain purpose functional their and co-associations promoter underlie that nisms populations across cell frequency at though cell types, low in specialized especially ways can colocalize, or bodies, Polycomb factories with transcription poised associate to found are genes repressed factories transcription called transcription, of sites in cluster units transcription active that suggest approaches ( open regulation gene of major understanding the a in question is units transcription different chroma between contacts long-range tin and states expression gene between link The chromatin the homotypic predictions. or simulations distinct investigate Gene Inês de Santiago Mariano Barbieri extended Active and poised promoter states drive folding of the nature structural & molecular biology molecular & structural nature Received 5 December 2016; accepted 27 March 2017; published online 24 April 2017; Epigenetic Epigenetic Regulation and Chromatin Architecture Group, Berlin Institute for Medical Systems Biology, Max Delbrück Centre for Molecular Medicine, Berlin, Mouse ES cells lack lamin A and have lower levels of heterochro of levels lower have and A lamin lack cells ES Mouse

binding

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2 activation Dipartimento Dipartimento di Fisica, Università di Napoli Federico II, and INFN Napoli, Complesso di Universitario Monte Sant’Angelo, Naples, Italy.

folding

folding sites Institute Institute for Biology, zu Humboldt-Universität Berlin, Berlin, Germany. the interactions

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CTCF the , , Miguel R Branco 1

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mouse Genome Genome Function Group, MRC London Institute of Medical Sciences (LMS) (previously MRC Clinical Sciences Centre), Imperial 3– 1 7

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CTCF. , HoxB , whereas Polycomb- whereas , [email protected] Polycomb-repressed Section Section of Virology, Department of Medicine, Imperial College London, Hammersmith Hospital Campus,

, , David Rueda , Elena , TorlaiElena Triglia

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5 locus, Single-cell predict Single Single Molecule Imaging Group, MRC London Institute of Medical Sciences (LMS), Imperial College

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© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. published classifications published within states promoter gene such region, and we mined published data sets of chromatin sites oroccupancy binding CTCF of position containing Hoxb13 95685813–96650449) (chr11: cluster the on centered region genomic 1-Mb a Weconsidered Chromatin RESULTS scale. at this folding explain not does alone occupancy CTCF whereas of of tens kilobases, scale length at genomic the locus of in roles folding the the but play simultaneous states separate promoter Polycomb-repressed and active the that Wefind cells. ES cell imaging using fluorescence the of scenarios folding different contribution of Polycomb), or (iii) CTCF occupancy. To discriminate the considering (without H3K4me3 and/or of RNAPII-S5p presence different promoter states (active or Polycomb-repressed), (ii) only the We considered of different scenarios chromatin contacts driven by (i) from polymer physics, the Strings & Binders Switch (SBS) and to dissect different possible folding scenarios, we use an approach cells: Polycomb-repressed or ES active mouse in activation transcriptional of states two in genes top s e l c i t r a Polycomb repressive marks as active, poised and silent states. silent and poised active, as marks repressive Polycomb Gip gene active for shown are Examples shown. also are profiles mRNA-seq and binding CTCF IP), (mock Control cells. ES mouse in H2AK119ub1 and H3K27me3 H3K4me3, RNAPII-S5p, for ChIP-seq by produced maps occupancy chromatin ( states. activation gene with relationship the and folding chromatin underlie that mechanisms 1 Figure

To explore the of mechanisms chromatin folding in the , all present within the the within present , all o logy. The

and a total of 28 genes ( genes 28 of total a and Classification of gene promoter states across the HoxB locus in mouse ES cells. ( cells. ES mouse in locus HoxB the across states promoter gene of Classification c a Phospho1 20 kb Phospho1

Abl3 states Abl3 HoxB What arethe Gngt2 interaction important Gngt2 sites?

locus is a complex locus containing closely spaced across AK133925 AK133925 B4gaint2 B4gaint2 HoxB 27 ,

36 the , 3 cluster. ( cluster. 3 7 . ( Igf2bp1

Igf2bp1 in situ HoxB Supplementary Table 1 Supplementary Fig. 1b Fig. HoxB chromatin fold? How does GipSnf8 Gip hybridization (FISH) in mouse

c Snf8 locus ) Classification of gene promoter states across the extended extended the across states promoter gene of ) Classification locus, we performed single- performed we locus, What arethe Atp5g1 mediators? , interaction Atp5g1 important c Ube2z Ube2z ). To define the genomic genomic the define To ).

Calcoco2 Calcoco2 in

mouse TtII6 TtII6 Hoxb1–Hoxb9

b H2AK119ub1 ES RNAPII-S5p b13 b13 H3K27me3 33– ). Control IP H3K4me3 HoxB HoxB mRNA CTCF AK078606 AK078606 advance online publication online advance cells 3 AK078566 5 AK078566 model. locus locus HoxB gene gene b9 b9 b8 b8 4,500 10 20 20 15 20 35 b7 , b7 b6 b6

b5 b5 AK002660 AK002860 b4 b4 SBS polymer model polymer SBS the of mechanisms folding the Todissect Modeling extended the of folding for the drivers as candidate locus, of the length 1-Mb full the in abundant are which sites, binding CTCF of list published a used shown previously as H3K4me3, ( 1a state Fig. Polycomb their of irrespective occupancy, RNAPII-S5p by marked states, promoter open of contribution sible 2 Table in classification (complete inactive as classified was Gip into or the active either states, promoter while classified poised of the example, (for H2AK119ub1 or H3K27me3 marks at TSS regions Polycomb-repressive but not the mRNA of expression one of least at presence of and the by RNAPII-S5p defined was state promoter (poised) repressed the example, (for marks Polycomb-repressive of site absence start the and (TSS) transcription the on centered window 2-kb a within S5p which chromatin conformation is shaped by chromatin interactions interactions chromatin by shaped is conformation chromatin which in a The SBS simulates scenario model determinants. molecular sible b3 active Classify genepromotersaccordingto b3 Atp5g1 Active The active promoter state was defined by the presence of RNAPII- by presence the defined was state promoter active The 5 kb b2 b2 gene was not found occupied by RNAPII-S5p or H3K27me3 and or by H3K27me3 RNAPII-S5p was not gene occupied found Atp5g1 b1 b1 and – ). As an additional folding scenario, we considered a pos a considered we scenario, folding additional an As ). c poised ). RNAPII-S5p-positive promoters are often positive for for positive often are promoters RNAPII-S5p-positive ).

different gene, gene, Atp5g1 a ) Our study focuses on understanding the molecular molecular the understanding on focuses study ) Our chromatinstates Hoxb9 b Skap1 HoxB Skap1 ) Gene activation states can be identified using using identified be can states activation ) Gene Fig. 1b Fig. , Polycomb-repressed gene gene , Polycomb-repressed

33 folding , , nature structural & molecular biology molecular & structural nature locus ( locus Fig. 1b Fig. , Poised Hoxb9 3 HoxB 5 kb 5 , to simulate the effects of the different pos different the of effects the simulate to c ), as described previously ), as described locus according to RNAPII occupancy and and occupancy RNAPII to according locus

conformations , Supplementary Fig. 1d Fig. Supplementary c ). We found that 27 promoters can be be can promoters 27 We that ). found 3 (see Online Methods). Finally, we we Finally, Methods). Online (see Silent HoxB Gip 5 kb Hoxb9

the locus, we used the the used we locus, and silent gene gene silent and Snx11 Snx1

3 HoxB C 1 . . The Polycomb- Supplementary Supplementary Supplementary Supplementary ). Cbx1 bx 1

locus - -

© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. extent a simplified model of chromatin folding, based what only on to the bio testing on focused was aim our at resolutions, folding genomic lower chromatin explain to particular in considered, be could general fully sense, this in and are, number) coordination lattice or length polymer example, (for modynamic phases do not depend on specific choices of model details 2 Fig. and red binders for green beads polymer of binding of energy the varying tions, concentra binder fixed at obtained diagram phase The Methods). and constant Boltzmann energies binding for diagram E phase system the derived and range relevant logically molecules, binding active and poised of concentrations the We varied system. model such of states mational sites. binding six bind to allowed was binder simulations repeated where binding sites later could only be bound we by a single binder while each comparison, a As binders. six to up by simultaneously bound be could beads polymer in interacting case which the discussed first we sites, binding polymer the of valency factory transcription a in ist of magnitude of the number that of is polymerases to suggested coex order in same the lattice), on cubic the number coordination (the six the of a maximum up to of sites, binding multiple bind sites can and color same binding to bind to allowed only is factor binding Each ( factors binding ‘poised’ and ‘active’ molecules: promoted Brownian of is kinds different binding two by Specific effects. volume excluded for except interact not do they and state, (gray) inert the in are beads polymer other the All state red) of activation. (or Polycomb-repressed, poised across the locus ( genes of activation of state the reflect promoters gene of coordinates Polycomb- repressor complexes and RNAPII with associated abundantly most are that regions gene the are promoters because regions), coding whole not site. We classified binding sites according to promoter states only (and to the of same kind affinity binding of with beads the co-associations ( ers were defined by whether they contained active and/or poised promot above. described states chromatin the from chosen were factors binding and sites binding of types and locations The conditions. ary bound periodic with lattice square a on polymer the simulated we efficiency, For computational bead. same the within states promoter to of was avoid chosen of bead each the different presence with genes The genomic length size finite effects. tail to polymer counterbalance on each added were beads inert 12 kb; 7.6 to corresponds bead ymer times, we defined a polymer with a total of 152 beads, where each pol simulation computer reasonable Toachieve Methods). Online (see chain bead polymer self-avoiding a as SBS the within modeled cells of events calization chromatin of nuclei fly and frequency mammalian in FISH by the measured contacts and distance genomic between ship data Hi-C describe successfully been to has used model previously SBS The factors. molecular binding cognate with at genomic locations specific (or binding sites; nature structural & molecular biology molecular & structural nature derive to sufficient be could promoters, gene of functionality logical R Using computer simulations, we explored the equilibrium confor equilibrium the explored we simulations, Using computer sites binding potential the which in polymers considered We first the containing region genomic 1-Mb-long The = = 39 Fig. 1 Fig. E ). It is well established in polymer physics that the system ther system the that physics in polymer ). It established is well , 4 G 0 = 4 = , and more and , c ) and in which contacts could only be mediated through through mediated be only could contacts which in and ) k B E T R , a value in the range of real transcription-factor- real of range the in value a , Fig. 2 44 and and i. 2 Fig. , 4 41 4 5 Xist 3 ( , 4 . . In such models, polymer containing beads the a 2 Fig. 2 Fig. E . ), ), namely active (green) state of activation and a loci during X inactivation in female mouse mouse female in inactivation X during loci G , green and red free beads, respectively). respectively). beads, free red and green , , has a similar structure ( structure similar a has , T the temperature in Kelvin (see Online Online (see Kelvin in temperature the 2 b , 2 , see Online Methods). Here, Here, Methods). Online see , 2 4 . To investigate the effects of binding binding of effects To the . investigate 6 . Although additional complexities complexities additional Although . c R and and Fig. Fig. 2

c advance online publication online advance G , in a broad, bio broad, a in , a HoxB Supplementary Supplementary 3 , , red and green) 3 , the relation the , locus was was locus 33 , k 3 8 B , colo , is the the is 33 , 3 5 ------

tice units, units, tice lat three than more no by distant are they that probability (the sites polymer of probability contact pair-wise the represent that matrices contact produced we phase, each in modeled polymers of ensemble structures with the intervening gray polymer loopingbeads out. bindingbinding sites to specific andtheir form two separate compact drive folding. Finally, in phase 4, both red and green beads compete for ule, corresponding to an active in model onlywhich active promoters glob a form beads green the and open are beads red the 3, phase In conditions, only Polycomb-repressed promoters drivethese SAWIn state.the a in locus independently folding. float promoters) (active sites ously to form a ‘red’ compact globule, while the green polymer binding polymers. In phase 2, only the red (Polycomb-like) beads fold spontanedom folded conformations, for self-avoiding (SAW) as expected walk Fig. 2 four corresponding conformational and classes phases ( thermodynamic distinct four identifies energies binding the of modeling Polymer The locus. complex a of mechanisms folding larger-scale stand under help to experimentally tested be could that scenarios folding detected with optimal sensitivity by cryoFISH sensitivity optimal with detected of a and are region, properties 1-Mb to they folding study the general resolution spatial high provide these as polymer), the in beads four cover ~30- to 40-kb genomic regions (corresponding to approximately validate chromatin4C-seq contacts gling and correlation with their chromosome translocations factories scription or to tran with active used previously poised study gene associations architecture cellular of preservation optimal with FISH combines that approach the across distances interchromatin HoxB the determine to out set we the of architecture real To the explore Testing candidate the of positioning cells. ES mouse the in regions interacting visualizing by experimentally ( folding of patterns distinct clearly have models different the with obtained matrices contact seven The in state a and which the form beads interacting a state globule, compact conformation open SAW a expected: as phases, two only has is only one of type binding factor, such that the system phase diagram site locations ( binding CTCF by driven is folding which Polycomb in model a of and presence), (irrespective H3K4me3 and RNAPII of presence the with associated states promoter to open are linked only sites binding for scenarios other the ing 3, and 2 phase of mixture (50:50 states red-only and green-only the in folded of polymers mixtures we in cell, same the considered alleles different of chromatin across oreffect contacts cell populations heterogeneous observed. also are effects bystander 4); (phase contacts homotypic red 2), 3) only (phase (phase green or a combination of and green red only reflect which phases, three other in the emerge patterns contact different strikingly Conversely, contacts. random of chances higher have sites neighboring only as signature, diagonal a only has matrix ( matrices tact To help visualize the contact patterns and the frequency across the across and frequency the Topatterns contact the help visualize We also considered three additional cases. First, to explore the the explore to First, cases. additional three considered also We

model ; see also ref. locus in single cells using cryoFISH, a high-resolution imaging imaging a high-resolution cryoFISH, using cells in single locus

the Fig. 2 Fig.

0 conformational 4 model ). Each thermodynamic phase gives rise to distinct con distinct to rise gives phase thermodynamic Each ). 8 , , followed by confocal microscopy Supplementary Fig. Supplementary 1c Fig. 2 Fig. d ). We also implemented polymer models represent models polymer implemented We also ). 3 1

3 , predictions 2 ). In phase 1, polymers are fully open and have ran c , 4 ). In phase 1, the fully open phase, the contact contact the phase, open fully the 1, phase In ). , to study chromosome volume and intermin and volume chromosome study to , HoxB HoxB

classes locus in different concentrations or concentrations different in locus folding: a model in which polymer polymer which in model a folding: 5 0

Fig. 2c Fig. . . We fosmid selected probes that and , d HoxB ). ). In the two latter cases, there

their Fig. 2 , locus in mouse ES cells, cells, ES mouse in locus d ), which can be tested tested be can which ), 4

contact . b 4 9 and . . This approach was s e l c i t r a Supplementary

matrices 4 8 , , and to 4 7 ------.

© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. ( radius gyration polymer’s of measure by phases different the in shown is state open SAW random the to respect with locus, the of degree compaction in increase relative the Here binders. of concentration by defined phases, thermodynamics its to correspond which model, the of classes architectural stable the identifies ( folding. locus of conformations equilibrium the identify physics polymer from simulations Carlo Monte respectively. binders, (red) ‘poised’ and (green) ‘active’ bind can sites binding poised and Active (gray). silent or (red) poised (green), active as classified states promoter gene represent that sites binding with informed model, polymer bead-chain SBS an into resolution a 7.6-kb at mapped is locus 2 Figure s e l c i t r a contacts. CTCF-dependent by driven folding and RNAPII-S5p, of presence the exclusively considering promoters, open by d

) Additional contact probability patterns from control models: a 50:50 mixed population of phase 2 and phase 3 models, folding driven driven folding 3 models, phase 2 and phase of population mixed a 50:50 models: control from patterns probability contact ) Additional

The SBS polymer model predicts folding of the HoxB locus under different biological scenarios. ( scenarios. biological different under locus HoxB the of folding predicts model polymer SBS The d c b a poised sites attheHoxB 2. Definebinding promoter states driven byactivepromoters 1. Classify Phase 3– locus and Phase 1–open Snf8 active Phase 2and3mixture HoxB13 green Phase 1 closed Snf8 HoxB1 R g / R g 0 . . ( Snx11 Snx11 c ) Different contact probability patterns are identified in the four thermodynamic phases. phases. thermodynamic four the in identified are patterns probability contact ) Different R g HoxB13 0.6 0.8 1.0 / R

0 g

Open (RNAPll-S5p log(

0.01 promoters C R Phase 2 ), (% 0.05 ) advance online publication online advance HoxB1 + ) concentration of red and binders 3. Vary green CTCF bindingsites 0.05 Phase 3 log( C 0.01 G ), (% ) nature structural & molecular biology molecular & structural nature 4. MonteCarlosimulations Driven byactiveandpoisedpromoters 0 a Phase 4– ) The 1-Mb extended extended 1-Mb ) The driven bypoisedpromoters Contact probability Phase 2– Phase green 0.5 b ) The system phase diagram diagram phase system ) The 4 red and closed red

1.0 closed HoxB

© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. FISH experiments (performed on ~150-nm-thick cryosections cut cut cryosections ~150-nm-thick on (performed experiments FISH ( H3K4me3 and marks Polycomb of devoid the genes ( active of pair distant the with not but regions, AK078606 between contacts in result would Fig. 3b only based on by promoter RNAPII-S5p ( occupancy lower with intensity ( but contacts of patterns similar observe would we case latter the in as 3), and 2 phases of mixture a from 4 phase example, (for separate or simultaneous are contacts red–red and green–green whether distinguish also can probes of set same The 4. and 2 phases genes, 1 or 2 ( phases in not but 4 and 3 phases in probability high with other each genes, (green) containing active regions two the promoters, poised and active consider profiles ( contact different have that kb, ~30 covering each regions, of models folding extended the different seven the discriminate to required be folding. of mechanisms distinct very to correspond that maps contact different clearly have testing experimental for chosen regions genomic five the whereas (red), genes poised as classified of promoters on centered regions ~35-kb covering probes FISH 3 Figure nature structural & molecular biology molecular & structural nature in generated models polymer 3D the and orientations) random in Supplementary Fig. 3c Fig. Supplementary Fig. 3 Fig. To identify the minimal number of genomic regions that would would that regions genomic of number minimal the identify To To allow direct comparisons between distances derived from To cryo derived distances between comparisons direct allow Skap1 Hoxb1–Hoxb2 a Supplementary Fig. 3a Fig. Supplementary ). ). Finally, polymer folding driven by CTCF-binding sites alone

, , Genomic regions chosen for testing model predictions of of predictions model testing for chosen regions Genomic Supplementary Table 3 Table Supplementary coding region which contains CTCF binding sites but is is but sites binding CTCF contains which region coding Supplementary Fig. 3b Supplementary (poised), and to a lesser extent extent and to a lesser (poised), a b Hoxb13 Hoxb13 Control Control Hoxb Snx1 Hoxb Snx1 Snf8 Snf8 HoxB 1 1 1 1 Snf8 Snf8 driven byactivepromoters locus, we identified five evenly spaced genomic genomic spaced evenly five identified we locus, Hoxb13 Hoxb13 Phase 1–open and Snf8–Ube2z green Phase 3– ). The intervening control region lies within within lies region control intervening ). The FISH probes Hoxb Hoxb Hoxb13–AK078606 closed 1 1 Ctrl Ctrl ). The regions containing poised (red) (red) poised ). containing The regions ). ). It a would model distinguish also ). For example, in the models that that models the in example, For ). Snf8–Ube2z Snx1 Snx11 and and Snf8 1 Snf8 Ube2z Snf8 Snx11–Cbx1, Hoxb13 Control Hoxb13 Hoxb Control Snx1 Hoxb Snx1 Snf8 Snf8 Hoxb1–Hoxb2 and and 1 1 1 1 ~150 kb Supplementary Fig. 4 Fig. Supplementary driven byactiveandpoised Snf8 , would contact only in only contact , would Snf8 (active) and and (active) driven bypoisedpromoter green Snx11

Phase 2– advance online publication online advance Hoxb13 Hoxb13 promoters and Phase 4– Supplementary Supplementary would contact contact would are classified as active (green). ( (green). active as classified are Hoxb13 AK07860 Hoxb Hoxb b13 red Snx11–Cbx1 and control red 1 1 closed Snf8 closed Ctrl Hoxb13– Ctrl 6 HoxB ~170 kb , , Sn×11 Snx1 Hoxb1 s locus folding by FISH in ES cells. ( cells. ES in FISH by folding locus 1 ).

, , Hoxb13 between other probe combinations. probe other between ( regions genomic active two the regions genomic ( poised two the marking probes two the between ( images the improve to nm) (~150 cryosections thin uses it as analyses these for relevant only by separated are kb ( 150–730 regions five The probes. five the of nations combi pairwise ten all for cryoFISH performed we modeling, mer ES mouse of five the nucleus between the cells in distances physical the determine To ES Measuring the of folding of predictions different make scenarios different the to according eled mod polymers from generated mini-matrices the all expected, As of the projection the on 3D sidering distance the plane of the section. con models, polymer the in region genomic each of mass of centers the between distances interlocus the determined we sections, the of plane the in measurement distance and cryosections of imaging the To simulate Methods). Online (see experiments cryoFISH the from those to equivalent thickness with polymers simulated our ‘slices’ through virtual random on cutting by process models polymer of sectioning ensemble the the simulated we conditions, different the Hoxb13 Control Hoxb13 Hoxb Hoxb1 Control Snx1 Hoxb Snx1 Snf8 Hoxb1–Hoxb2 Snf8 b2

1 1 cells 1 1 b1 Snf8 Snf8 and and Open (RNAPll-S5p

CTCF bindingsites Fig. 4 Fig. by Hoxb13 Hoxb13

promoters Snx11 cryoFISH chromatin b ~240 kb Fig. 4 Fig. z ) Mini-contact matrices obtained from modeled polymers of polymers modeled from obtained matrices ) Mini-contact -axis diffraction limit. Visual inspection of cryoFISH cryoFISH of inspection Visual limit. diffraction -axis a Hoxb1 Hoxb1 and ) suggested higher levels of homotypic colocalization colocalization of homotypic levels higher ) suggested HoxB genes and at a control region. region. a control at and genes a Ctrl Ctrl Hoxb13–AK078606 ), making high-resolution cryoFISH particularly particularly cryoFISH high-resolution ), making + ) locus ( locus

HoxB Snx11 contacts Snx11 a Control ) Scheme of the positions of the selected selected the of positions the of ) Scheme locus regions selected based on poly on based selected regions locus Fig. 3 Fig. Hoxb13 Contro Hoxb1 Snx1 Snf8

Snf8–Ube2z within 1 l b Snf8 Phase 2and3mixture ). ~170 kb ) ) and the two probes marking Hoxb13

1.0 0 0.5 the

probability Hoxb HoxB and and Contact Hoxb1 1 Snx11 Ctrl Snx11 Snx11–Cbx1 s e l c i t r a

locus and and Cbx1 Snx1 1 Hoxb13

mouse ) than than ) are are

- - - -

© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. regions of the the of regions poised and active between interactions homotypic distinct that suggesting ensembles, polymer slicing by obtained model SBS the of 4 prediction phase ( experiments. FISH independent two least at Hoxb13 right, (top regions left, (top poised between distances average The probes. of pairs different between distances physical 2 Bars, (blue). TOTO-3 with counterstained were acids Nucleic image. the in signal probe each represent to chosen pseudo-color the indicate names gene at pointing arrowheads Colored states. promoter (red) poised and (green) active represent names gene around over probes ( model. SBS the by predicted as promoters, 4 Figure contact between these two regions that persists across the cycling cycling of stable reports with agrees the observation This cells. of ES across population persists that regions two these between contact close spatially and frequent a identifying alleles, of (~75%) majority Hoxb2 ( The centers distances of mass of mean signals between the two their poised regions by ( shown also as ( region control the including than distributions of narrower pairs from different classes (heterotypicstrikingly pairs) or are pairs pairs) poised–poised or active–active (homotypic class same the from regions genomic between distances probe signal (see Online Methods). We found that the distributions of thickness. section in result differences small from may experiments cryoFISH between detection in variability The small compaction. locus and potentially efficiency labeling their 7 was detection average the to difference experiment; FISH same the within quency 5b Fig. 14 average duced give balanced frequency of locus detection per nuclear slice (on distances for all probe pairs, data set of per probe distances ~200 interlocus pair interlocus (~2000 the contain not do and most nuclear profiles random to orientation, the sectioning thin due expected, As profiles). nuclear ~20,000 of total a to (adding profiles nuclear 1,535–2,587 from images collected we pairs, probe ( HoxB the within region genomic each of micros position the confocal identified and by copy images cryoFISH of set data large a collected s e l c i t r a Polycomb-repressed domains between associations Supplementary Fig. 5a Fig. Supplementary

We then measured the distances between the centers of mass of each the of description To quantitative a obtain c a Hoxb13 Control Hoxb Snx11 Snf8 locus using a semi-automated macro implemented in ImageJ ImageJ in implemented macro semi-automated a using locus and and , , ). Red and green signals were detected with proportional fre proportional with detected were signals green and Red ). Hoxb13 1

n Imaging the extended extended the Imaging Snf8 = 149) or loci relative to control (bottom right, right, (bottom control to relative loci or = 149) HoxB ± Hoxb13–AK078606 Hoxb13 3%, as expected in ~150-nm sections; sections; ~150-nm in expected as 3%, HoxB CryoFISH Hoxb1 Hoxb locus. Cryosections (150 nm thick) were hybridized with probes specific to to specific probes with hybridized were thick) nm (150 Cryosections locus. ± HoxB Snf8 4%, most likely due to differences in probe length, length, probe in differences to due likely most 4%, 1 locus are a major folding feature. folding a major are locus Ctrl and and Snx11 locus ( locus Snf8 Driven byactiveandpoisedpromoters , see Online Methods). For each of the ten ten the of each For Methods). Online see , Snx11, n Snx11, Snf8 Hoxb13 Control Hoxb Snx11 Phase 4- Snf8 Supplementary Supplementary Fig. 5b ~150 kb HoxB

1 Supplemetary Fig. 5a Fig. Supplemetary Snx11 Snf8 Fig. 4 Fig. ) are found within 100 nm in the vast vast the in nm 100 within found are ) Hoxb13

Hoxb1 locus by cryoFISH identifies a folding pattern dependent on homotypic contacts between active and poised poised and active between contacts homotypic on dependent pattern a folding identifies cryoFISH by locus = 292) are significantly shorter (<150 nm) than between other pairs of loci (for example, bottom left, left, bottom example, (for loci of pairs other between than nm) (<150 shorter significantly are = 292) green 3 ~170 kb b Hoxb1 and and and Hoxb1 a Hoxb1 Ctrl c ) CryoFISH images of of images ) CryoFISH upeetr Fg 5b Fig. Supplementary ) The contact mini-matrix determined experimentally by cryoFISH imaging is equivalent to the polymer polymer the to equivalent is imaging cryoFISH by experimentally determined mini-matrix contact ) The red Snx1 Supplementary Fig. 6 Fig. Supplementary S 3 HoxB closed 1 ~240 kb nf 8 ). The probes pro locus folding, we folding, locus 5

1 Supplementary Supplementary ), resulting in a in resulting ), . Colocalization . Colocalization Control Snx11 0 0.5 1.0 Contro ~170 kb advance online publication online advance probability and control; control; and Contac l Hoxb1– HoxB Hoxb1 Snx1 1 t 3 locus folding. Top, schematic of the genomic positioning of the cryoFISH cryoFISH the of positioning genomic the of Top, schematic folding. locus ). ). ), ), - - -

the same polymer. We found that the pattern of folding is the same, same, the is folding of pattern the that We polymer. found same the green and corresponding red respectively, contact their beads, within gene poised can beads (red) Polycomb and and (green) active both where promoters, active between contacts simultaneous considers match 4 phase exactly of matrices which the model, the experimental that found we Strikingly, models. polymer 3D slicing by produced in shown matrices the with comparable directly is matrix a colocalization threshold of 105 (using nm, colocalization probe FISH of frequency the calculating by the seven model we predictions, produced a contact matrix cryoFISH the of folding the To compare Comparing colocalize. not nm do that locus same the 250 within for regions detected above distances from distinguishable promptly are which zation distances below ~100 nm, for subregions within the regions along genome. the Welinear spaced colocali critical identify closely between even distances, genomic fine-mapping in cryoFISH within the extended kilobasepairs and active Polycomb-repressed promoter regions, across hundreds of of compartmentalization spatial the identify results These nm. >250 by separated (65–90%) the majority vast of the with nm, <100 (10–25%) at distances proportion small a only find we regions, of pairs remaining the all for contrast, In alleles. both for the often in population, cells ES of majority vast the in colocalize regions active two are close equally (~80 nm) and frequent indicating (~75%), that these contact of frequency and distance physical their sequence, genomic genes ( active covering probes two the Remarkably, although nm. of age ~70 between nm regions 100 genomic two the of distance average an yielding FISH 3D by cells between n b = 76), most often found at >250 nm. Distances were collected from from collected were Distances nm. >250 at found often most = 76), Frequency (%) Frequency (%) 10 20 30 20 40 60 80 0 0 Snf8 ~150 kb 0 0 Snf8–Ube2z Hoxb13 Hoxb1 Hoxb13 50 50 ~

170 kb

Distance (nm polymer Distance (nm) 100 100 Hoxb1 Hoxb1 and and 150 150 and

, , Hoxb9 nature structural & molecular biology molecular & structural nature Hoxb13 Hoxb1 200 200

) models Snx11–Cbx1 5 2 , compatible with our findings of an aver an of findings our with compatible , >250 had previously been shown in mouse ES ES mouse in shown been previously had >250 and and , , Snx11

HoxB with Hoxb13

HoxB Frequency (%) Frequency (%) µ 80 20 40 60 Fig. Fig. 4 10 20 30 ) ) are separated by 730 kb in linear , , m. ( m. 0

locus observed in ES cells with with cells ES in observed locus 0 locus Snf8 Snf8 0 0 locus, and locus, show the power of , , b Hoxb13 n ) Histograms of inter-locus inter-locus of ) Histograms c and control. Colored boxes boxes Colored control. and ; ; see Online Methods). The = 290) or active promoter promoter active or = 290) 50 50

distances Distance (nm Distance (nm) 100 100 ~400 kb ~ 730 kb 150 150

Control 200 ) 200 HoxB Snf8 Figure 3 Figure >250 Snx11 >250 and and locus, b - -

© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. model simulations with the same red and green binder-binding site site binder-binding green and red same the with simulations model SBS the repeated we region, DNA binding other one only with loops region binding DNA each whereby regimen, folding relaxed more a by obtained be could properties same the genes. whether test active To further contain that kb 730 by separated regions distant two between contacts long-range identifies and in particular population, cell the across allele each within contacts abundant of formation the the within identified of folding mechanisms The chromatin Polymer areeight cases) seldom found with interdistances below 200 nm. other (all regions of pairs non-homotypic Conversely, nm. 100 than of single cells in the heterogeneous ES cell population at distances lower active and genes poised simultaneously occur across the vast majority between homotypic interactions distinct which in scenario a support in the fine-tuning folding at shorter Our analyses hence length scales. the FISH probes Weused). do not exclude a role for CTCF occupancy the folding scale of tens of kilobases (~30–40 kb, the sequence length of experimental measurements across the the all capture to sufficient oversimplicity,is its of regardless model, polymer SBS gene active/poised the that suggest together, results Takenour condensation). chromatin local or enhancers of presence as (such regions these within contacts internal additional or crowding SAW their byfolding altering locus the tuning local include behavior of details the binding sites will need to be considered in the future to explain further homotypic genomic regions, suggesting that other folding properties or the between than heterotypic between distances the forrecapitulated details of the distance distributions measured by cryoFISH ( are less well experimentally observed not were that rations distance distributions become bimodal and include much larger sepa the 3), and 2 phases (50:50, polymers separate in occur interactions the mixture model is considered, where active–active or poised–poised tions for phase 4 ( determined in ES cell nuclei are in good agreement with model predic and ( 4 phase in polymers of ensemble the for predicted tributions dis distance the with experimentally observed distributions distance able to explain further details of folding, we compared the entire locus cells. same the within alleles separate even or cells separate in not contacts are independent by and explained nuclei cell ES within predominant highly are contacts HoxB recently explored those as areas, gene-poor in roles deterministic have may other the in seen as folding, overall the to contribute across the gene-dense experimentally detected contacts of matrices the explain to unable particularly are ( alone sites by CTCF-binding mediated the Contacts cases. two between contacts of pattern identical overall an of irrespective ( experimentally identified contacts of chromatin frequency ( cells different separate or in alleles occur and polymer same the in exclusive mutually are in regions active regions and which contacts between poised between experimental FISH data. In particular, the mixed population scenario, the with incompatible are modeling polymer by obtained scenarios active of ~75% alternative Conversely, the nm. 105 within found with regions and poised similar, is contacts of frequency the also and nature structural & molecular biology molecular & structural nature by most at bound be to bead polymer each allowing but affinities To explore to what extent the predictions of our simple models were Supplementary Fig. 7 Fig. Supplementary locus suggest that homotypic (active–active and poised–poised) 1

conformations 5 . Our detailed analyses of physical distances across the the across distances physical of analyses detailed Our .

contacts HoxB Fig. locus folding. Featureslocus that can play a role in fine- 5 and

Fig. 3 Fig. depend ). We). distributions found distance that the Supplementary Supplementary Fig. 7 b ), is not compatible with the high high the with compatible not is ),

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highly correlated with the corresponding matrix from the lattice lattice the from matrix corresponding the with correlated is 4 highly phase in matrix contact the particular, In modeling. on-lattice the MD produce simulations a to phase diagram Montesimilar Carlo explained in Online Methods). As expected from statistical mechanics, 5.2 to (equal folding 4 phase generate to previously energies, interaction same the with potential, Lennard–Jones and is binders by given beads truncated an attractive, chain between interactions the that We lattice. considered cubic a in to as Montean and sites simulations alternative beads, binding Carlo polymer between potentials on force-field based approach modeling simulations (MD) dynamics molecular To investigate the further MD contacts. local multiple of abundance of the folding the that suggest ( six is multiplicity binder when 4 phase with correlation best the identifies modeling SBS the and cryoFISH by identified proximities locus the between analysis A correlation results. by experimental disproved a together, scenario and containing regions of the two active distant contact, multiplicity maintained at six). Perhaps more strikingly, in these conditions of low is multiplicity the when of polymers >75% with contrast (in of ~50% Hoxb1–Hoxb2 the the contacts between two Polycomb-repressed regions containing ( binder one interactions, although free fitting parameters were not used to optimize optimize to used not were parameters fitting free although interactions, homotypic 4) (phase simultaneous with polymers SBS of ensemble an using physics polymer by and cryoFISH single-cell from experimentally obtained were distributions distance Similar distances. inter-locus of distributions the to extends model interaction homotypic SBS the and 5 Figure 148, 292), 292), 148, 290), cryoFISH, 151; mix, 2–3 phase 137; 4, (phase for measurements distance of Number 3 polymers). phase 2 and phase of mixture (50:50 interactions homotypic green–green or red–red only with polymers of a mixture from produced polymers SBS of ensemble an from obtained profile the from different is experimentally obtained distances of profile The cases. other the and homotypic between the that Note nm). 150 above (~70% broader much are that distances probes control and heterotypic with contrast in nm, <150 are distances homotypic the of (~90%) Most match. the Probability Probability 0.1 0.2 0.3 0.4 0.2 0.4 0.6

Snx11, simulations 0 0 0 0

Heterotypic interactions Agreement between cryoFISH imaging of the the of imaging cryoFISH between Agreement

0.15 0.05 Hoxb1 separated by 730 kb (or 97 polymer beads), do not come come not do beads), polymer 97 (or kb 730 by separated Supplementary Fig. 8a Fig. Supplementary and Model: phase 0.30 Distance 0.10

– reproduce Snf8 Hoxb13–AK078606 0.45 0.15 (74, 90, 100) and and 100) 90, (74, ( µ m) Hoxb1–Hoxb13 M 4 HoxB 0.60 Hoxb1–Snf8 Supplementary Fig. 8b Fig. Supplementary 0.20

Homotypic interactions simultaneous odel: phase2–3mix(50:50) HoxB >0.75 polymer conformation, we conformation, polymer employed >0.25 locus is best explained by a high by a high explained best is locus 0.2 0.4 0.6 0.1 0.2 0.3 0.4 ). Interestingly, we found that that found we Interestingly, ). 0 0 are still detected at a frequency 0 0 34 Snx11 Control cases , 5 x 0.15 3 0.05

-axis scales are different different are scales -axis , which are an off-lattice off-lattice an are which , homotypic –Control (70, 91, 76). 91, (70, –Control E 0.30 Distance 0.10 R Snf8 s e l c i t r a and and HoxB – k g b 0.45 ). These results results These ). T 0.15 CryoFISH Hoxb1 – ( , more details details more , locus locus

µ Snx11 E contacts m) G 0.60 , as found found as , Snf8–Snx1 0.20 Snx11–Ctrl – Hoxb13 (126, (126, >0.75 >0.25 Snf8 1

© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. To further explore the nature of the homotypic contacts involving involving contacts homotypic the of nature the explore further To transcription Homotypic ( binders red and green between contacts homotypic simultaneous capture also tions shows that MD simula which a across range of parameters, matrices We of contact the MD simulations. parameters similar field obtained force- the in changes to robust are findings our whether tested also 97%, of correlation (Pearson’s model s e l c i t r a active or Polycomb-repressed regions, we tested whether the observed 23, 120 and 78, 56 and 99. and 56 78, and 120 23, and 25 74, and 42 right: to left replicates, technical for measurements of Number RNAPII-S2p. not but EZH2, with colocalization with coincides regions (poised) Polycomb-repressed of colocalization Conversely, S2p. and promoters active containing regions genomic of Colocalization adjacent). are or overlap not do (signals ‘separated’ or 1 pixel) least at by overlap signals (fluorescence ‘colocalized’ as scored were Loci respectively. complexes, repressor Polycomb and factories transcription Hoxb1 bar, 2 Scale (blue). DAPI with counterstained were acids Nucleic positions. contact locus indicate Arrowheads EZH2. or RNAPII-S2p to relative imaged were ( genes Polycomb-repressive or respectively) red, and green pseudo-colored ( genes active of position The blue). pseudo-colored ( RNAPII-S2p for specific antibodies with immunolabeled indirectly were thick) nm (~150 cryosections cryoFISH, Before immuno-cryoFISH. by determined was EZH2 component Polycomb or RNAPII (S2p) active with ( complexes. Polycomb and RNAPII-S2p with colocalization respective their with coincide 6 Figure c a e c a

, , d c pseudo-colored blue) and the catalytic Polycomb subunit EZH2 ( EZH2 subunit Polycomb catalytic the and blue) pseudo-colored : : Snf11 Hoxb13 and and

Homotypic interactions between active and poised genes genes poised and active between interactions Homotypic preferentially coincides with colocalization with RNAPII- with colocalization with coincides preferentially

Hoxb13 % Triple colocalization µ and and

20 40 60 80 contacts m. ( m. 0

factories Hoxb13 e Hoxb1 Supplementary Fig. 10 Fig. Supplementary ) Frequency of association of of association of ) Frequency with active RNAPII-S2p and EZH2, markers of active active of markers EZH2, and RNAPII-S2p active with Snf8 S2p S2p a Hoxb1 Snx11

– coincide Snf8 , pseudo-colored green and red, respectively) respectively) red, and green , pseudo-colored S2p S2p d

) The association of active and poised genes genes poised and active of association ) The or –

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Hoxb1 and and Snx11 EZH2 EZH2 Snx8 advance online publication online advance or Snx11

. We ). b , d

, - calize with each other, they are more likely to colocalize with with colocalize to likely more ( EZH2 are with not but they RNAPII-S2p other, each with calize 6c Fig. Cbx1 ( active interacting two the with EZH2 or PRC2; of subunit catalytic (the EZH2 of the active (Ser2 phosphorylated) RNAPII or with Polycomb subunit immunodetection with regions FISH of genomic or the active poised with RNAPII-S2p and respectively EZH2, genes Polycomb-repressed and active single of colocalization shown had cells ES mouse in work immuno-cryoFISH previous Our Polycomb bodies called (also components or Polycomb-complex factories transcription called (also RNAPII of form active the ing of or regions with nuclear active domains contain Polycomb-repressed association simultaneous the with coincide contacts homotypic locus. the of parts different together pulls which force the are regions enrichment CTCF to correspond sites binding the which in a model and classification); promoters poised and active (no binders all with affinity similar has promoter each which in promoters, gene between interactions heterotypic on based a model binders; with affinity have promoters poised only or active only that as such considered, is promoters of type one only among interaction partial which in models two architecture; locus the shape sites chromatin of distance genomic relative by determined collisions random only which in case, non-interacting the cryoFISH: by cells single in measured positioning locus with consistent ( FISH. by tested the of view linear right, Below model. 4 SBS phase the of conformation 3D the of snapshot Right, promoters. gene poised and active between interactions homotypic on uniquely based model a polymer by scale, 30-kb the at explained, ( 7 Figure a b a ) The folding properties of the the of properties folding ) The We performed immuno-cryoFISH by triple labeling RNAPII-S2p RNAPII-S2p labeling triple by immuno-cryoFISH Weperformed Red–red andgreen–green homotypic interactions , , Heterotypic interaction Fig. 6a Fig. Non-interacting case ,

Poised RNAPII Polycomb body Active RNAPII d Active Model representing representing Model TSS ) ein. e on ta we to cie ein colo regions active two when that found We regions. Poised TSS , b b ) or poised ( poised or ) Control ) The alternative folding mechanisms tested were not not were tested mechanisms folding alternative ) The

nature structural & molecular biology molecular & structural nature Nonspecific binders HoxB Hoxb1–Hoxb2 Snx11 HoxB Snf8 locus folding in mouse ES cells. cells. ES mouse in folding locus Snf8 binding locus in mouse ES cells are are cells ES mouse in locus CTCF Hoxb13 sites Fig. 6 Fig. Only red–redoronlygreen–green 3 HoxB . . Therefore, we combined cryo Fig. Fig. homotypic interactions e locus predicted by the the by predicted locus Hoxb1 ). In contrast, colocalized colocalized contrast, In ). 6 and and Snf8–Ube2z CTCF-based ). interactions HoxB Hoxb13–AK078606 Hoxb13 Control CTCF locus regions regions locus and and Control Hoxb1 Snx11 Snx11–

3 , 6 2 ). ). - - ) ,

© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. the the Helmholtz Foundation (A.P., M.B., E.T.T.), and the InstituteBerlin of Health supported by the Medical Research Council, UK (A.P., S.Q.X., I.d.S., M.R.B., D.R.), ESC-OS25 cell line (MRC Human Unit,Genetic Edinburgh, UK). The work was and C. Ribeiro de Almeida for help and advice, and W. Bickmore for providing the We thank K.J. Morris for help growing the mouse ES cells, E. Brookes, Beagrie R.A. t the in available are references, and codes accession statements of including Methods, and data availability any associated M states. chromatin and architecture chromatin between relationship the of understanding high-resolution imaging of specific loci opens the and way towardgenome a linear deeper the across analyses feature epigenetic modeling, of combination the that shows study present the More organization. generally, chromatin via part complex regulatory a playing interactions long-range with domains, local segregated of series a than complex more is folding chromatin that suggests This space. 3D in closest the necessarily not are regions genomic tiguous states functional across the chromosome fiber and confirms that con loci. specific of folding the to regions genomic neighboring the of contributions additional including whole chromosomes, will be required to further understand regions, genomic broader considering studies Future folding. locus on regions the of proximal most the impact main the identifies study ( distances genomic for ( law power a as separation genomic with decays ability that probcontact the average Considering itself. chromatin pairwise which is approximately one order of magnitude longer than the the cluster on centered region genomic 1-Mb the considered observations recent with consistent results, FISH with patible binding sites CTCF involving contacts of formation through organization locus ( of PRC2, EZH2. We also investigated other folding possible scenarios component enzymatic the with or RNAPII of form phosphorylated) (Ser2 the with active colocalization with respectively, coincide, genes Polycomb-repressed or active between between proximity the spatial ( genes Polycomb-repressed between and genes active between alone, state activation promoter on dependent contacts, homotypic of kinds two of formation with consistent were results cryoFISH Single-cell folding. locus of mechanisms different distinguish could that regions genomic critical we identify to able were modeling, polymer of help the With level. single-cell the at ing the on HoxB centered region gene-dense 1-Mb a of organization 3D the to contacts gene-promoter of contribution the investigated we Here, DISCUSSION and respectively. bodies, Polycomb active and between factories transcription at occur interactions promoters Polycomb-repressed homotypic that suggest results these Furthermore, interactions). (heterotypic states different with Polycomb-repressed or active regions between of are contacts low genes) with favored, abundances between interactions (homotypic states epigenetic similar with regions chromatin among interactions ( RNAPII-S2p not but EZH2 with ciated co-asso found often more are regions (poised) Polycomb-repressed nature structural & molecular biology molecular & structural nature (A.P., M.B., M.N.). This work was supported by grants to M.N. from CINECA A h Fig. 7 Fig. c e ethods Our Our approach shows that 3D chromatin folding is driven by specific k

p n a locus by combining polymer physics simulations with imag with simulations physics polymer combining by locus owl p b e ), in particular the hypothesis that CTCF alone could drive drive could alone CTCF that hypothesis the particular in ), r . ed 11– g ments 1 4 . . However, CTCF-driven folding alone was not com s i te .–0 b range Mb 0.5–10 the in ) Fig. 6 Fig. Fig. 7 Fig. e

). This confirms that that confirms This ). a advance online publication online advance ). We showed that that showed We ). in silico in o n 3 3 l i , our present present our , HoxB n e P

v ( polymer polymer e s cluster, r ) ~ ~ ) s 1 i 0 o . We . n s −1

o - - - - - ) f

6. 5. 4. 3. 2. 1. claims jurisdictional in published maps and affiliations. institutional r at online available is information permissions and Reprints The authors declare no competing interests.financial authors the revised manuscript. analysis pipeline. A.P., M.N., M.B., S.Q.X., A.M.C. and S.B. wrote the paper, and all advice and co-mentored the work of S.Q.X. with A.P. M.R.B. developed the image I.d.S. and E.T.T. performed the bioinformatics analyses. D.R. provided conceptual modeling analysis. S.Q.X. performed the wet-lab experiments and image analysis. A.P. and M.N. the designed project. M.B., A.M.C., and S.B. performed the polymer CINECA, and ISCRA ID M.N. HP10CYFPS5. also acknowledges computer resources from INFN, 28. 27. 26. 25. 24. 23. 22. 21. 20. 19. 18. 17. 16. 15. 14. 13. 12. 11. 10. 9. 8. 7. C AUTH e O

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© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. tially as described previously CryoFISH on ultrathin cryosections (immuno-cryoFISH) was performed essen immunofluorescenceRNAPII-S2pdetectionof combinedEZH2or DNA with DNA-FISH combined with immunofluorescence of RNAPII or EZH2. with VectaShield mounted were(Vector coverslips Laboratories), before immediately before PBS imaging. in washed and PBS in Tween-20 2 with stained were NucleiInvitrogen). 1:1,000, h, (1 andAlexaFluor488-conjugated antibodies raised donkeyin against mouse IgG antibodies against FITC IgG (1 h, 1:500, Jackson ImmunoResearch Laboratories)1:1,000,Invitrogen).h, (1 Probes FITCwithlabeledwere mousewithdetected AlexaFluor555-conjugatedwith antibodiesdonkeyagainst inraised sheepIgGand Roche) 1:200, (2h, fragments Fab anti-digoxigenin sheep with detected Jackson ImmunoResearch Laboratories). Probes labeled with digoxigenin were Cyanine3-conjugated donkey antibodies raised against rabbit IgG (1 h, 1:1,000, andInvitrogen) 1:500, h, (2 antibodies anti-rhodamine rabbit with amplified was probes rhodamine-labeled of signal The gelatin. fish-skin 0.1% and BSA, 0.5% NaCl, 2.6% containing VectorLaboratories) 7.8, (pH solution blocking casein- with min) (30 incubated were Sections min). 10 °C, (42 SSC 4× in 20 Tween-0.1% and min), 30 over 3× °C, (60 SSC 0.1× min), 25 over 3× °C, (42 (30 min) at 37 °C. Beforehybridization, probes were denatured (10 min) at 70°Cand reannealed 1 mide (Sigma), 2× SSC, 10% dextran sulfate, 50 mM phosphate buffer (pH 7.0), forma deionized 50% contained mixturesHybridization moisth. 48 over a chamber in °C 37 at out carried was Hybridization dehydrated. and SSC, 2× formamide, deionized 70% in °C) 80 min, (10 denatured each), min 3 series, dehydratedHCl,100% to M ethanol(500.1 in with min) treated (10SSC), 2× 250 with °C) 37 h, (1 incubatedwere Cryosections with 0.2% Triton X-100 and 0.2% saponin in PBS, and then washed (3×) in PBS. (15 min) with 20 mM glycine in PBS, rinsed (3×) in PBS, permeabilized (10 min)Snx11 Hoxb13– follows: before described as essentially thickness) nm (150 cryosections fresh on directly performed was CryoFISH Fluorescence columns (Bio–Rad). chromatographyP-30 MicroBioSpin using nucleotides unincorporated from purified and (Roche), translation nick by tetramethyl-rhodamine-5-dUTP or Table( 4 primers specific using PCR by confirmed was probes mid and control(WI1-2809P17135) including BACPACUSA; from (California,probes Resources Probes. loops (~2 mm diameter), and transferred onto glass coverslips for cryoFISH. ultracryomicrotome (Leica), captured in sucrose drops on metal bacteriological Tokuyasu cryosectionsnitrogen. liquid (150 in nmfrozen andin PBS thickness)in sucrose wereM 2.1 cut in usingh) (2 an embedded UltraCutwere UCT 52 provideswhich optimalpreservation subcellular oforganization formaldehyde in 250 mM HEPES-NaOH (pH 7.6, 10 min and 2 h, respectively), Briefly, cells were fixed in 4% and then 8% EM grade freshly depolymerized para tested for mycoplasma contamination. mg/ml Hygromycin (Roche) as described previously mia inhibitory factor (LIF; Chemicon, Millipore, Chandler’s Ford, UK) and 0.1 Invitrogen), 50 Gibco, from (all pyruvate sodium mM 1 (NEAA), acids amino non-essential in GMEM-BHK21 supplemented with 10% FCS, 2 mM as previously described Cell culture. ONLINE doi: antibodies primary murine with immunolabeled were thick) nm (~140–150 µ Post-hybridization washes were as follows: 50% formamide in 2× SSC SSC 2× in formamide 50% follows: as were washes Post-hybridization Cells were fixed and processed for cryosectioning as described previously g/ 10.1038/nsmb.3402 µ . Briefly, cryosections on glass coverslips were rinsed (3×) in PBS, incubated l Cot1 DNA, 2 ). Probes were labeled with digoxigenin-11-dUTP, fluorescein-12-dUTP Genomic regions within the the within regions Genomic Snf8–Hoxb13 Control, Snf8

METHODS Mouse ES-OS25 cells (kindly provided by W. Bickmore) were grown (WI1-696E14), n situ in µ M Hoxb13–Snx11, Hoxb1– β -mercaptoethanol, 1000 U/ml of human recombinant leuke µ , yrdzto o utahn roetos (cryoFISH). cryosections ultrathin on hybridization g/ Snf8–Hoxb1, Snf8– 5 3 µ . Briefly, cells were grown on 0.1% gelatin-coated flasks l salmon sperm DNA and 2–4 3 , 4 HoxB13 48 . Briefly, for , Snx11 5 6 for cohybridizationforof (WI1-2076M19), HoxB (WI1-1523P20). The specificity of fos of specificity The (WI1-1523P20). Control Control Figure 6a locus were detected using fosmid using detected were locus , Snf8–Snx11, Hoxb13–Hoxb1 , Hoxb1 54 µ Supplementary TableSupplementary3 – g/ml RNase A (Sigma, in (Sigma, A RNase g/ml , 5 d µ 5 µ , ultrathin cryosections L . mESCs were routinely Hoxb1 l nick-translated probe. M TOTO-3 in 0.05% in TOTO-3 M – -glutamine, 1% MEM HoxB Snx11, (WI1-376M13), Supplementary Supplementary locus pairs, as pairs, locus 5 and Control 7 . Cell pellets Cell . Cryo- 4 , 48 , 5 6 ), – - - - - - . ,

allele (homologous and heterologous) distances. inter- pairwise measure all tocoordinates centerused themass andoftheirof profiles based on the counterstain, loci present within each profilemass estimationwere and distance identifiedcalculation. Briefly, after segmentation of nuclear Fig. 6 collected sequentially to prevent fluorescence bleed-through. were channelsdifferent from Images disk. Airy 1 to equivalent pinhole using 405-nm diode, and argon (488 nm), HeNe (543 nm) and HeNe (633 nm) lasers, scanning microscope (Leica TCS SP5, 63× oil objective, NA 1.4) equippedanalysis. with image a and Microscopy coverslip was sub-sampled to indicate variability. in the case of imaging. Measurements were obtained from two independent coverslips, except 1 with stainedwerenuclei and nocomplexesduring FISH. CryoFISHwas thenperformed above, asdescribed freshly-depolymerized PFA in PBS (2h), before DNA-FISH, to preserve immu 8% with fixed were cryosections immunolabeling,After shown). not cells, ES RNAPII in western blots or by omission of primary antibodies to show lack of detection treatmentof phosphorylated with alkaline phosphatase (2 h, 37 °C, 0.5 U/ Invitrogen). The specificity of antibodies to phosphorylated with RNAPIIAlexaFluor680-conjugated was donkey testedantibodies against by mouse Ig (1 h, 1:500, or with mouse antibody against EZH2 (2 h, 1:50, BD BioSciences) and detected specifically against RNAPII phosphorylated on S2 (2 h, H5, IgM, 1:2000, Covance) locus are also positiveforareH3K4me3, accordingalso previously locus a to published list the in RNAPII-S5p for positivepromoters the of 89% that We observe and classification considered for this study is shown in promotersof list complete The version. genome mm9 the of database UCSC coordinates,as previously described integrating levels of ChIP-seq enrichment within 2-kb windows centered on TSS the oncentered region genomic 1-Mb a within genes Weconsidered text). main (see H2AK119ub1 and H3K27me3 RNAPII-S5p, of absence or presence the to according silent classification. Promoter sites were obtained in ES-E14 (ref. H3K27me3 data sets were produced in ESC-V6.5 (ref. obtained in ES-OS25 cells, grown in the same conditions as in the present study. e ( tool LiftOver UCSC the using 2007) (July mm9 version to (Feb.2006) mm8 versiongenome mouse fromconverted coordinateswere tions. TopHat was run with default parameter settings for single-end reads using TopHat the mouse genome mm9 and the UCSC mm9 Known Gene GTF annotation file were merged after mapping and removal of replicated reads. When reads originated from multiple sequencing runs of the same library, they the 95th percentile of the frequency distribution of each data set, were removed. the same genomic location) that occur more often than a threshold, computed aswith default parameter settings. Replicated reads (i.e. identical reads, aligned to (ref. Bowtie2 usingassembly, 2007)(mm9 Julygenome mouse the data ( Browser mRNA-seq data sets used are shown in sets. data occupancy chromatin of Mapping overlap, including adjacent signals). ‘separated’or pixel) 1 least atoverlap‘colocalized’ (signals as Hoxb13 stretched raw images in Adobe Photoshop. The association betweencontrast first we EZH2, subunit Polycomb or RNAPII-S2p active containing d u Formeasurementsinter-locus of distances( aa es o RAI-5, 2K1u1 mc-P n mN-e were mRNA-seq and mock-IP H2AK119ub1, RNAPII-S5p, for sets Data Coordinates of CTCF binding sites were previously published For mRNA-seq, sequenced reads from a published data set were aligned against For ChIP-seq, sequenced reads from published data sets were realigned against domains nuclear and regions genomic between colocalization Tomeasure / c g Fig. 1 ), we used a supervised automatic algorithm for locus detection, center of i - or 5 b 9 i ( n Snf8 b h / h t ). Snf8-Snx11 t 6 g p 1 L and v2.0.8 to allow the detection of reads on exon-exon splice junc : / i / f g t O e n Snx11 v o e m r ). Most (93%) regions were successfully converted. HoxB 5 nature structural & molecular biology molecular & structural nature colocalization with RNAPII-S2p, where data from one e 3 and immunofluorescence . u loci with RNAPII-S2p or EZH2 ( rmtr wr casfe a atv, osd or poised active, as classified were Promoters c s locus. The binary classification was obtained by obtained was classification binary The locus. c . e µ d g/ u Images were acquired on a confocal laser- confocal a on acquired were Images 36 ) was used to display single-gene sequencing µ 3 l DAPI in 0.05% Tween-20 in PBS, beforeTween-20PBS, 0.05% DAPIin in l , . TSS coordinates .TSS were retrieved from the 3 Supplementary Table 1 7 ) ( Supplementary Table 1 Figs. 4 Figs. h pbihd hPsq and ChIP-seq published The 5 8 (confirmed here in mouse µ , Supplementary Table 2 3

l, New England BioLabs) 5 6 , and and , ) while CTCF binding h Fig. 6 t t p 4 . UCSC Genome Supplementary (signals do not do (signals : 3 / 7 / . The genomic g e ). e ) was scored n Hoxb1 6 o 0 m ) v2.0.5, ) e HoxB . u 6 and 2 c . s c 3 - - . . .

© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. those of the model requires a specific approach whereby we consider analogous with sections nuclear cryoFISH in probes of relativedistance and frequencies 3 i.e., spacing, three-lattice of in Contact and frequency matrices. single MC steps SAWthe (see state polymerconfigurations obtained frominitial noninteracting configurations in independent-equilibrium-500 to up produced we concentrationvalue,factor mitted between nearest-neighbor particles per onlyinteractionsare non-breakingconstraints maintained. Chemical are polymerand siteoccupancy singlelattice, while the nearestonneighbor cella tions ensuring the compaction of the polymer. gies used in the previous model ( have up to six bonds with different binding sites. We used the onlysame one (versusbinding six in the previous version),ener while binders keep their ability to malnumber of bonds that binding sites can form with the binders is limited to 1 ( al. et tude of the typical number of polymerases in a transcription factory (see Pombo ingmoleculeshave bindinga multiplicity valueorderofofathe six, of magni concentration, The fractions of binding molecules per lattice binding site is relatedenergies (where to their molar E In particular, in the main text and c concentrationsgenes,activeandthe ‘poised’of and‘active’ binding molecules, corresponding to the in order to avoid finite size effects on the conformation of chainthe total portionlength also includes of12 inert beadsthe added atchain each end of the polymer excluded volume effects (and chain length integrity constraints, see below). The factors. All other (gray) beads are inert, as they have no interactions apart from analogously, active genes (green in are attachment sites for the ‘poised’ binding factors (also visualized in red) and, the chain. In the SBS simulation conditions chosen, poised genes (red in separated by less than 7,600 bp, they are represented as contiguous beads along ( assemblygenome mm9 the from are coordinates used The Transcription (TSS). gene Sites Start R radius was determined from the radii of nuclear slices used for cryoFISH using the size of the mouse genome, of the average nuclear radius of cell nucleus. Thus, we have within a lattice polymer bead as equal to the average density of chromatin in the scale of In order to compare cryoFISH data with our simulation results, we must set the a single bead in a cubic lattice cell of unitary edge size, the chain is made of bead chain on a cubic lattice of unitary edge size, model (SBS) Switch Binders & Strings the employed we mm9), genome mouse kb, the around Montesimulations. Carlo via model SBS or proximity to other genes. not listed in the RefSeq list or their promoters were not classified due to overlap ( ( positive. H3K4me3 as sified promoters,positive RNAPII-S5p two Only nature structural & molecular biology molecular & structural nature kb 30 roughly are probes FISH As model. polymer our through cut sections Hoxb5os 4833417C18Rik R R = and Supplementary Fig. 8a = The model was tested by Metropolis Monte Carlo (MC) computersimula (MC) MonteMetropolisCarlo by tested was model The We explored the system behavior varying the affinity, Beads associated with each gene within the Figure 2 Figure π E , 1999 (ref. 1999 , 64 × < G 33 , 6 c = 4 d 5 , G 3 . Diffusing molecules and polymer beads randomly move from one to 0 r ) did not have a classification in the published list 5 . These values are varied in a broad, biologically relevant rangerelevantbiologically broad, a in varied are values These . . This was accomplished by estimating the chromatin average density > / 2, where < where a chromatin filament is represented as a self-avoiding polymer k c HoxB B , report the probability of finding a bead pair within a distance a within pair bead a finding of probability the report , T c , a value which is in the range of real transcription factor binding 64 m k ), 2 B , : cluster in mouse ES cells (chr11: 95685813-96650449, 960 960 95685813-96650449, (chr11: cells ES mouse in cluster 6 ), and references therein). The SBS model with multiplicity with model SBS The therein). references and ), is the Boltzmann constant and c 5 AK078606 Supplementary TableSupplementary2 Fig. 2 Fig. ~ . N HoxB c = 152 beads. The length of each bead ( m r > is the average radii of nuclear slices. d b , 0 b ). Each run is fully equilibrated with up to 5 × 10 × 5 to up equilibratedwith fully is run Each ). 3 region. d N , e 0 , A ( ~( Phospho1 d f G (where ) is a variant of the above model where the maxi Gm11538 0 R = . The comparison between the co-segregation the between comparison The . , we obtain s E 0 Fig. 2 The model Contact Matrices, as those shown R Figures 2 / 5.0 G = ) 1/3 E N µ G a , , R ) are attachment sites for ‘active’ binding m derived from nuclear profiles and of = 4 A ), Abi3 . Considering the order of magnitude is the Avogadro number). Our bind 33 ToDNAregionselected the model AK078566 – d ). In the few cases where TSSs are TSSs where cases few the In ). k , 3 Hoxb2 5 0 B 5 , , HoxB ~0.035 . For each red and green binding T and Gngt2 ), and a concentration of 0.01% d 0 T

(ref. 7 and region are positioned at the the temperature in Kelvin). , we refer to the case where , , ( d µ Snf8 E 0 Gm53 m. The average nuclear 6 , contains R Hoxb8 3 and ). In the present case, 3 , , , because they were Ube2z ), and ), s E 0 , were not clas not were , ) is 7,600 bases; G , of poised and s , , 0 base pairs. AK133925 AK002860 Fig. 2 33 , 3 a 12 5 ------) .

upon reasonable request. Dataavailability. upon request. therein. Guidance and information on any custom computer codes publicationscitedprevious are in availableand Methods Online the in details all in trated availability. Code between contact matrices. Statistical testing. in the literature (see backbone,without noticeable changes majorthe features contactofthe matrix polymer the of potential interaction FENE the of range andamplitude the as such range, broadcomparatively a in model polymer MD our of parameters E of values higher with found also were 4 phase ofresults similar found binders ( sitesbinding cognate the their and of energy interaction the varied we First, changes. of types two is robust to changes in the force-field parameters. In particular, we implemented eters. The structure of the contact matrix in the different thermodynamic phases we sample configurations each 10 τ set, in dimensionless units, equal to 0.5 (ref. coefficient friction The case. MC the to equal volumeconditionsand ary softwareLAMMPS the in Verletimplementedalgorithm, the via integrated is equation Langevin for Monte Carlo lattice simulations and cut-off distance equal to 2 to equal distance cut-off andsimulations lattice MonteCarlo for energies, interaction with potential, Lennard-Jones truncated attractive, an by given is Lennard-Jones potential 30 (refs. nonlinear elastic (FENE) spring finite the use we chain, polymer the of beads consecutive two between bonds mass,same the tookand the diameter and beads binders of our polymer systems Langevinthe equation. obey We set field potentials developed in classical studies of polymer physicstions were performed exploiting polymer modeling approaches based on force- Molecular dynamics computer simulations. some 11, where distances >1 differenthomologousin loci chromosomesdistancesbetween excluding ofby oFISH ( independently for each pair of polymer regions. was random-sliced once for each pair of polymer regions; slicing was performed independentEach polymerconfigurationcryosections. real in one used the to Inabovethe procedure, thicknesssection correspondingofnm,a 150 we used co-segregationprobabilitythe as distanceoffindingthis within3 measuredtheir we Then longitudinalsection.projected alongthe the planeof distance their computed we section, the in were mass of centers both where cases the Incenterscontainedmass.itof notwo, checkedwhetheror oneand ‘section’ (having the same thickness used in the experiments) at a random angle fosmid probe ( (3 beads 3 of length same the with regions polymer consider we model SBS the in long, 54. = 0.012 (ref. G k We also tested the robustness of our results against different modeling param For comparison between distances obtained from the model and from cry fromand model the fromobtaineddistances Forcomparison between (compare

iln N, oiou, . Yn, .. Sih A & af M Nra tmn of timing Normal M. Raff, & A. Smith, Q.L., Ying, C., Jolicoeur, N., Billon, ES cells. ES oligodendrocyte development from genetically engineered, lineage-selectable mouse B Supplementary Fig. 10c T 23 / σ Fig. , 2 2 E (refs. 4 s R ), and the elastic constant 0 and 5

µ J. Cell Sci. Cell J.  σ and m, approximately the expected diameter of the territory of chromo Supplementary Fig. 10a Fig. Supplementary Supplementary Table 3 2 of the beads and binders equal to 1 (in MD dimensionless units) 23 kb) centered on the TSS of the specific gene included in the includedgenein specific the of centeredTSS kb)the 23on 23 3 23 HoxB E ). The system is then equilibrated up to 5 × 10 , 2 Supplementary Fig. 7 Thedata that support findingsthe ofstudy this are available Pearson’s correlation coefficient was used to estimate similarity G , 4 2 h agrtm o or utm optr oe ae illus are codes computer custom our of algorithms The 2 . , spanning the range from 0 to 8 4 5 ). The excluded volume effects are modeled by a repulsive . The system evolves in a cubic box with periodic bound systemperiodic Thecubicwithevolvesbox .a in is located.

115 23 m , 2 , 3657–3665 (2002). 3657–3665 , 4 , for bead and bindersand set and forbead , – . The interaction between chain beads and binders e ). This is consistent with previous results reported 2 4 E 6 . The FENE length constant, R timesteps. and K ). Then we cut through the model polymer , which gives the spring strength, is set to , ), we corrected for the small contribution b E ). Second, we varied other force-field other varied we Second, ). G , 5.2 and 6.0 and 5.2 , 2 Molecular dynamics (MD) simula 4 ), and the integration time step is k b T , in the same range used doi: k b m T 10.1038/nsmb.3402 = 1. To1. = the model , respectively) and respectively) , 23 R 8 , 2 timesteps, and 0 4 , is set to 1.6 . The polymer d 0 = 105 nm. 105 = σ 23 , 2 E 4 R . The . and ζ is σ ------

Kent, W.J. RNA of forms phosphorylated different of Distribution A. Pombo, & S.Q. Xie, Guillot, P.V., Xie,S.Q.,Hollinshead,M.&Pombo,A.Fixation-inducedredistribution Oct-3/4 hybridization of situ in fluorescence CryoFISH: A. expression Pombo, & L.M. Lavitas, S.Q., Quantitative Xie, A.G. Smith, & J. Miyazaki, H., Niwa, (2002). study. ultrastructural an cells: human in bodies PML and Cajal to relation in II polymerase Res. cells. human of nucleus the in II polymerase RNA hyperphosphorylated of cryosections. ultrathin on (2000). 372–376 cells. ES of self-renewal or dedifferentiation differentiation, defines 10.1038/nsmb.3402

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